Transformant and process for producing polyester by using the same

ABSTRACT

The present invention relates to a gene coding for a copolyester-synthesizing enzyme, a microorganism which utilizes said gene for the fermentative synthesis of a polyester, and a method of producing a polyester with the aid of said microorganism. More particularly, the present invention relates to a gene which functions in a host organism and is related to synthesize, by an enzyme, a plastic-like polymer degradable under the action of microorganisms in the natural environment (the soil, river or sea), a transformant derived from said host organism by transformation with said gene and having an improved ability to fermentatively synthesize a plastic-like polymer, and a method of producing a copolyester with the aid of said transformant.

TECHNICAL FIELD

[0001] The present invention relates to a gene coding for acopolyester-synthesizing enzyme, a microorganism which utilizes saidgene for the fermentative synthesis of a polyester, and a method ofproducing a polyester with the aid of said microorganism. Moreparticularly, the present invention relates to a gene which functions ina host organism and is related to synthesize, by an enzyme, aplastic-like polymer degradable under the action of microorganisms inthe natural environment (the soil, river or sea), a transformant derivedfrom said host organism by transformation with said gene and having animproved ability to fermentatively synthesize a plastic-like polymer,and a method of producing a copolyester with the aid of saidtransformant.

BACKGROUND ART

[0002] It is already known that many kinds of microorganisms accumulatepolyesters as the energy-storing substance intracellularly. Arepresentative example is a homopolymer of 3-hydroxybutyric acid(hereinafter referred to briefly as 3HB), namely poly-3-hydroxybutyricacid (hereinafter referred to briefly as P(3HB)), which was firstdiscovered in Bacillus megaterium in 1925. Since P(3HB) is athermoplastic polymer biodegradable in the natural environment, it hasattracted attention as an eco-friendly plastic. However, because of itshigh crystallinity, P(3HB) is so hard and brittle that it has foundapplication so far only in a limited assortment of practical uses andmuch research work has been undertaken to correct for the drawback.

[0003] As results of such researches, Japanese Kokai PublicationSho-57-150393 and Japanese Kokai Publication Sho-59-220192 disclose theproduction technology for a copolymer of 3-hydroxybutyric acid (3HB) and3-hydroxyvaleric acid (3HV) (hereinafter referred to briefly asP(3HB-co-3HV)). Compared with P(3HB), this P (3HB-co-3HV) is so flexiblethat it was initially expected to find application in a broader range ofend uses. Actually, however, P(3HB-co-3HV) responds poorly to a gain inthe molar fraction of 3HV so that particularly the flexibility requiredof film and the like cannot be improved, thus limiting its scope of useto rigid moldings such as shampoo bottles and throw-away razor handles.

[0004] Recently, studies have been undertaken on the binary copolyesterof 3HB and 3-hydroxyhexanoic acid (hereinafter referred to briefly as3HH) (the copolyester will hereinafter be referred to briefly asP(3HB-co-3HH)) and the technology of producing it. For example, suchstudies have been described in Japanese Kokai Publication Hei-5-93049and Japanese Kokai Publication Hei-7-265065. The technology described inthe above patent literature for the production of P(3HB-co-3HH)comprises fermentative production from a fatty acid, e.g. oleic acid, oran oil or fat, e.g. olive oil, with the aid of a soil-isolated strain ofAeromonas caviae. Properties of P(3HB-co-3HH) have also been studied [Y.Doi, S. Kitamura, H. Abe: Macromolecules 28, 4822-4823 (1995)]. In thisreport referred to above, Aeromonas caviae is cultured using a fattyacid of 12 or more carbon atoms as the sole carbon source tofermentatively produce P(3HB-co-3HH) with a 3HH fraction of 11 to 19 mol%. This P(3HB-co-3HH) undergoes a gradual transition from a hard,brittle one to a flexible one with an increasing molar fraction of 3HHand has been found to show flexibility surpassing that of P(3HB-co-3HV).However, this production method is poor in productivity with a celloutput of 4 g/L and a polymer content of 30% and, therefore,explorations were made for methods of higher productivity for commercialexploitation.

[0005] A PHA (polyhydroxyalkanoic acid)-synthase gene was cloned from aP(3HB-co-3HH)-producible strain of Aeromonas caviae [T. Fukui, Y. Doi:J. Bacteriol, Vol. 179, No. 15, 4821-4830 (1997), Japanese KokaiPublication Hei-10-108682]. When this gene was introduced into Ralstoniaeutropha (formerly, Alcaligenes eutrophus) and the production ofP(3HB-co-3HH) was carried out using the resulting transformant, the celloutput was 4 g/L and the polymer content was 30%. Further, by growingthis transformant on vegetable oil as the carbon source, a cell contentof 4 g/L with a polymer content of 80% could be accomplished [T. Fukuiet al.: Appl. Microbiol. Biotechnol. 49, 333 (1998)]. A process forproducing P(3HB-co-3HH) using bacteria, e.g. Escherichia coli, or plantsas hosts has also been described (WO 00/43525). However, there is nodisclosure of the productivity achieved by this production technology.

[0006] Since this polymer P(3HB-co-3HH) may have a broadly variablecharacteristic ranging from a rigid polymer to a flexible polymerdepending on the 3HH molar fraction, it can be expected to findapplication in a broad spectrum of uses from television housings and thelike, which require rigidity, to yarn, film and the like which requireflexibility. However, the productivity of said polymer is stillinvariably low in these production methods and none are considered fullysatisfactory for practical production methods of this polymer.

[0007] Recently, Leaf et al. have conducted studies on the production ofbiodegradable polyesters using a yeast, which is considered to elaborateacetyl CoA, the precursor of 3HB, with good efficiency as a producerorganism (Microbiology, Vol. 142, pp 1169-1180 (1996)). They introducedthe Ralstonia eutropha polyester synthase gene into Saccharomycescerevisiae, a kind of yeast, to construct a transformant and cultured itusing glucose as the carbon source, to thereby confirm the accumulationof P(3HB) (polymer content 0.5%). However, the polymer produced in thisstudy was P (3HB), which is hard and brittle.

[0008] It is known that yeasts are fast-growing, with high cellproductivity. The yeast cell attracted attention as the single cellprotein in the past and studies on the production of yeast cells for useas a feedstuff using n-paraffin as the carbon source, while theircomponent nucleic acids have been utilized as seasonings. Furthermore,since yeasts are considered to produce acetyl-CoA, which is a precursorof the polymer, with high efficiency, a high polymer productivity isexpected. Moreover, since the separation of cells from the culture brothis easy as compared with bacteria, it is possible to simplify thepolymer extraction and purification process. Therefore, a demand hasexisted for a process for producing P(3HB-co-3HH) having beneficialphysical properties with the aid of yeasts.

DISCLOSURE OF INVENTION

[0009] In light of the above state of the art, the present invention hasfor its object to provide a polyester synthesis-associated gene whichfunctions and can be expressed with good efficiency in a yeast, atransformant of the yeast as transformed with a gene expression cassettecomprising said gene, and a method of producing a polyester which isbiodegradable and has excellent physical properties, such asP(3HB-co-3HH), which comprises growing the transformant obtained.

[0010] After many investigations, the inventors of the present inventionconstructed a gene expression cassette by ligating a promoter and aterminator, both of which substantially function in a yeast, to at leastone enzyme gene related to the synthesis of a copolyester throughcopolymerization of 3-hydroxyalkanoic acids of the following generalformula (1) and can be substantially expressed in the yeast,

[0011] introduced said gene expression cassette into a yeast toconstruct a transformant,

[0012] and cultured said transformant, whereby a polyester, which is acopolymer of 3-hydroxyalkanoic acids of the following general formula(1), could be successfully harvested from the resulting culture.

[0013] in the formula, R represents an alkyl group.

[0014] The present invention, therefore, is concerned with atransformant

[0015] wherein at least one kind of gene expression cassettes comprisinga polyester synthesis-associated enzyme gene has been introduced into ayeast.

[0016] The present invention is further concerned with a method ofproducing a polyester using said transformant

[0017] which comprises growing said transformant

[0018] and harvesting the polyester from the resulting culture.

[0019] The term “substantially” as used herein means that referring tothe sequence of the polyester synthesis-associated gene and the genesequences such as a promoter and terminator, among others, which arenecessary for the construction of the gene expression cassette, thenucleotide sequences may have undergone mutation, such as deletion,substitution and/or insertion, as far as the functions of the gene andthe functions necessary for gene expression are retained.

[0020] Furthermore, the present invention is related to a polyestersynthesis-associated enzyme gene

[0021] which is modified from at least one gene code CTG to TTA, TTG,CTT, CTC or CTA.

[0022] The present invention is now described in detail.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic diagram showing the plasmid pSUT5 used as avector in Example 2 (a).

[0024]FIG. 2 is a schematic diagram showing the plasmid pUTA1 used as avector in Example 2 (b).

[0025]FIG. 3 is a schematic diagram showing the plasmid pSUT-phaJconstructed in Example 2 (a).

[0026]FIG. 4 is a schematic diagram showing the plasmid pSUT-PHA1constructed in Example 2 (a).

[0027]FIG. 5 is a schematic diagram showing the plasmid pSUT-PHA2constructed in Example 2 (a).

[0028]FIG. 6 is a schematic diagram showing the plasmid pUAL1constructed in Example 2 (b).

[0029]FIG. 7 is a schematic diagram showing the plasmid pUAL-ORF2constructed in Example 2 (b).

[0030]FIG. 8 is a schematic diagram showing the plasmid pUAL-ORF3constructed in Example 2 (b).

[0031]FIG. 9 is a schematic diagram showing the plasmid pUTA-ORF23constructed in Example 2 (b).

[0032]FIG. 10 is a plasmid construction diagram showing the procedure ofconstructing the plasmid according to Example 2 (a).

[0033]FIG. 11 is a plasmid construction diagram showing the procedure ofconstructing the plasmid according to Example 2 (b).

[0034]FIG. 12 shows the result of an analysis, by capillary gaschromatography, of the polyester produced in Example 3.

[0035]FIG. 13 is an NMR analysis chart of the polyester produced inExample 3.

[0036]FIG. 14 is an IR analysis chart of the polyester produced inExample 3.

[0037]FIG. 15 is an NMR analysis chart of the polyester produced inExample 4.

BEST MODE FOR CARRYING OUT THE INVENTION

[0038] (1) The Host

[0039] The yeast to be used is not particularly restricted but may beany of the yeasts belonging to the following genera and deposited withany of authoritative culture collections (such as IFO, ATCC, etc.):

[0040] Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya,Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera,Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus,Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus,Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium,Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella,Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus,Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces,Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces,Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia,Nakazawaea, Nematospora, Ogataea, Oosporidium, Pachysolen,Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula,Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia,Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces,Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus,Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces,Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon,Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia,Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus,Zygosaccharomyces, Zygowilliopsis, Zygozyma.

[0041] As the yeast used for the transformant according to the presentinvention, Candida malosa or Yarrowia lipolytica is preferred andCandida malosa is particularly preferred.

[0042] (2) The Polyester Synthesis-Associated Enzyme Gene

[0043] The polyester synthesis-associated enzyme gene is notparticularly restricted but is preferably one coding for an enzymederived from a bacterium. Specifically, preferred is an enzyme geneassociated with the synthesis of a copolyester of 3-hydroxyalkanoicacids of the above general formula (1), more preferred is an enzyme geneassociated with the synthesis of P(3HB-co-3HH) which is a copolyesterresulting from the copolymerization of 3-hydroxybutyric acid of thefollowing formula (2) and 3-hydroxyhexanoic acid of the followingformula (3).

[0044] The enzyme gene involved in the synthesis of a copolyester of3-hydroxyalkanoic acids of the above general formula (1) is notparticularly restricted but may for example be the polyester synthasegene described in Japanese Kokai Publication Hei-10-108682. As aspecific example of such polyester synthase gene, a PHA synthase genecan be mentioned. Furthermore, a polyester synthesis-associated enzymegene may be used in combination with the present polyester synthasegenes. Such enzyme genes include (R)-specific enoyl-CoA hydratase genewhich converts enoyl-CoA, an intermediate in the β-oxidation pathway, tothereby synthesize monomeric (R)-3-hydroxyacyl-CoA [T. Fukui et al.:FEMS Microbiology Letters, Vol. 170, 69-75 (1999)], β-ketothiolase genewhich dimerizes acetyl-CoA to monomeric 3-hydroxybutyryl-CoA, andNADPH-dependent acetoacetyl-CoA reductase gene [Peoples O P et al.: J.Biol. Chem., 264 (26) 15298-15303 (1989)].

[0045] Some species of said host yeast show abnormalities in thetranslating of genetic codes. For example, Candida cylindracea [Y.Kawaguchi et al., Nature 341, 164-166 (1989)] and Candida maltosa [H.Sugiyama et al., Yeast 11, 43-52 (1995)] are nonconventional yeasts inwhich the genetic code CTG is not translated into leucine but intoserine. When a polyester synthesis-associated enzyme gene is to beexpressed in such a yeast, genetic code translating abnormalities maytake place so that an enzyme having an amino acid sequence differentfrom that of the proper enzyme is produced at times. Therefore, thefunction of this enzyme is not fully exhibited.

[0046] Such a phenomenon can be avoided by using a gene constructed bymodifying the genetic code CTG contained in the gene beforehand to adifferent genetic code corresponding to leucine (TTA, TTG, CTT, CTC,CTA).

[0047] Analysis of the genetic codes of organisms inclusive of yeastsreveals that the frequency of usage of genetic codes varies widely withdifferent organisms. Thus, among a plurality of genetic codes specifyingthe same amino acid, the genetic code to be used vary with differentorganisms and it has been pointed out that the translation efficiency ofa gene consisting of genetic codes with high frequencies of use is high.For example, the GC contents of Aeromonas caviae PHA synthase gene and(R)-specific enoyl-CoA hydratase gene are 67.16% and 65.77%,respectively, but among the enzymes so far reported in Candida maltosa,the contents are 39.55% for phosphoglycerate kinase and 35.67% forALK2-A. Therefore, in order that a polyester synthesis-associated genemay be efficiently expressed in Candida maltosa, for instance, it ispreferable to use said gene in which said genetic code CTG is changed toa different genetic code specifying leucine and, at the same time, thegenetic codes are changed to those with high frequency of use.

[0048] The polyester synthesis-associated enzyme gene of the inventionmay be used as it is in a yeast which shows no translating abnormalityof the genetic codes. As an alternative, a modified gene as modified bychanging genetic codes to those used by the particular yeast with highfrequencies without alternation of the amino acid sequence may be used.In a yeast which shows translating abnormality of genetic codes, the CTGcodon of said enzyme gene may be changed to TTA, TTG, CTT, CTC or CTA,or a gene as modified by changing genetic codes to those used by theparticular yeast with high frequencies without alternation of the aminoacid sequence may be used. For example, when the host is Candidamaltosa, the gene identified in SEQ ID NO:3 or NO:4 can be used as thepolyester synthesis-associated enzyme gene of the invention. Thenucleotide sequence of this gene may have undergone mutation, such asdeletion, substitution, insertion and/or the like, provided that theresulting mutant gene produces the said polyester synthesis-associatedenzyme.

[0049] The polyester synthesized by said PHA synthase is a copolymer of3-hydroxyalkanoic acids of the above general formula (1), and can berepresented by the general formula (4) shown below. Preferred is thecopolyester P(3HB-co-3HH) obtainable by copolymerization of3-hydroxybutyric acid of the above formula (2) and 3-hydroxyhexanoicacid of the above formula (3) and can be represented by the followinggeneral formula (5).

[0050] (3) Construction of a Gene Expression Cassette

[0051] For the expression of a gene in a yeast, it is necessary toligate DNA sequences such as a promoter, UAS, etc. upstream of the5′-end of the gene and to ligate DNA sequences such as poly-A additionsignal, terminator, etc. downstream of the 3′-end of the gene. These DNAsequences maybe any arbitrary sequences that may function in the yeast.While the promoter includes sequences relevant to constitutiveexpression and sequences relevant to inducible expression, whicheverkind of such promoter sequence can be employed.

[0052] Moreover, in the transformant of the invention, the abovepromoter and terminator are preferably those which function in theorganism used for the production of a polyester.

[0053] Construction of a gene expression cassette to be used for theconstruction of a transformant according to the invention is nowdescribed, referring to (a) the case in which Yarrowia lipolytica isused as the host and (b) the case in which Candida maltosa is used asthe host as examples.

[0054] (a) When Yarrowia lipolytica is Used as the Host

[0055] When Yarrowia lipolytica is used as the host, the promoter andterminator to be used are preferably those derived from Yarrowialipolytica. More preferably, a promoter derived from Yarrowia lipolyticaALK3 and a terminator derived from Yarrowia lipolytica XRP2 are used.The DNA sequence of said promoter and/or terminator may be the DNAsequence derived by the deletion, substitution and/or addition of one ormore nucleotides as far as it may function in Yarrowia lipolytica.

[0056] The vector for use in the above construction may be any vectorwhich is a plasmid capable of autonomous replication in Escherichia coliand may have a region which will be autonomously replicated in theyeast. Such a vector capable of autonomous replication in the yeast willbe contained intracellularly. Moreover, the gene expression cassette canthen be integrated onto the chromosome. In Yarrowia lipolytica, theautonomously replicating pSAT4 and pSUT5 can be employed (Toshiya Iida:Studies on the n-alkane-inductive cytochrome P450 gene group of theyeast Yarrowia lipolytica, a doctorate dissertation thesis, TokyoUniversity Graduate School, 1997).

[0057] In the above yeast, the polyester synthesis-associated enzymegene is preferably a gene derived from Aeromonas caviae, and forexample, the A. caviae-derived PHA synthase gene (hereinafter referredto briefly as phaC) (SEQ ID NO:1) or the (R)-specific enoyl-CoAhydratase gene (hereinafter referred to briefly as phaJ) which convertsphaC and enoyl-CoA, which is an intermediate in the β-oxidation pathway,to the monomeric (R)-3-hydroxyacyl-CoA [T. Fukui et al., FEMSMicrobiology Letters, Vol. 170, 69-75 (1999)] (SEQ ID NO:2) can be usedwith advantage.

[0058] The promoter ALK3p (SEQ ID NO:5) (GenBank AB010390) of Yarrowialipolytica Alk3 gene can be ligated upstream of the 5′-end of each ofthese structural genes.

[0059] To prepare the restriction enzyme site necessary for linking thepromoter to the structural gene, a PCR technique can be utilized. Theprimer sequences used in the PCR are shown in SEQ ID NO:8 through NO:14.PCR conditions may be arbitrary as far as the objective gene fragmentcan be amplified.

[0060] As to the promoter, ALK3X with XbaI at 5′-end and NdeI at 3′-endand ALK3S with SacII at 5′-end and NdeI at 3′-end can be constructedfrom SEQ ID NO:8 and NO:9 and SEQ ID NO:9 and NO:10, respectively, usingSEQ ID NO:5 as a template. As to phac, a 100-bp (approx.) fragment withNdeI at 5′-end and PstI at 3′-end can be constructed from SEQ ID NO:11and NO:12 using SEQ ID NO:1 as a template. To this fragment is ligatedthe remaining 1700-bp (approx.) PstI-BamHI fragment to construct thefull-length phaC with NdeI at 5′-end and BamHI at 3′-end. As to phaJ,phaJ fragment with NdeI at 5′-end and KpnI at 3′-end can be constructedfrom SEQ ID NO:13 and NO:14 using SEQ ID NO:2 as a template. As to thevector, the plasmid vector pSUT5 (FIG. 1, SEQ ID NO:19) and the vectorpSUT6 obtainable by changing the NdeI site of pSUT5 to an XbaI site byusing a linker DNA shown in SEQ ID NO:20 can be used. To themulti-cloning site SacII, KpnI of pSUT6, ALK3S and phaJ can be ligatedto construct a plasmid pSUT-phaJ (FIG. 3). Then, to the multi-cloningsite XbaI, BamHI of pSUT5, ALK3X and phaC can be ligated to construct aplasmid pSUT-PHA1 (FIG. 4).

[0061] Further, ALK3S, phaJ and downstream terminator can be excised asa unit from the plasmid pSUT-phaJ using SacII and XbaI and ligated tothe SacII, XbaI site of plasmid pSUT-PHA1 to construct a plasmidpSUT-PHA2 (FIG. 5). In this manner, two kinds of plasmids forrecombination can be constructed.

[0062] By the above procedure, there can be constructed a geneexpression cassette for the production of a copolyester of3-hydroxyalkanoic acids of the above general formula (1) in the yeastYarrowia lipolytica.

[0063] (b) When Candida maltosa is Used as the Host

[0064] When Candida maltosa is used as the host, the promoter andterminator to be used are preferably those which function in Candidamaltosa, more preferably those derived from Candida maltosa. Still morepreferably, the promoter and terminator derived from Candida maltosaALK1 are used. The DNA sequence of said promoter and/or terminator maybe a DNA sequence derived by the deletion, substitution and/or additionof one or more bases only provided that the sequence may function inCandida maltosa.

[0065] The vector for use in the construction may be the same as the onereferred to above for the case (a). In Candida maltosa, the autonomouslyreplicating pUTU1 can be used [M. Ohkuma at al., J. Biol. Chem., Vol.273, 3948-3953 (1998)].

[0066] When Candida maltosa is used as the host, the polyestersynthesis-associated enzyme gene is preferably the gene coding for thesame amino acid sequence as the Aeromonas caviae-derived enzyme. Forexample, the gene coding for the same amino acid sequence as theAeromonas caviae-derived PHA synthase in Candida maltosa (this gene ishereinafter referred to briefly as ORF2, which is defined by SEQ IDNO:3), or the gene coding for the same amino acid sequence, in Candidamaltosa, as ORF2 and the (R)-specific enoyl-CoA hydratase which convertsenoyl-CoA, which is an intermediate in the β-oxidation pathway, to themonomeric (R)-3-hydroxyacyl-CoA (this gene is hereinafter referred tobriefly as ORF3, which is defined by SEQ ID NO:4) [T. Fukui et al., FEMSMicrobiology Letters, Vol. 170, 69-75 (1999)], can be used withadvantage.

[0067] The promoter ALK1p (SEQ ID NO: 6) and terminator ALK1t (SEQ IDNO:7) (GenBank D00481) [M. Takagi et al., Agric. Biol. Chem., Vol. 5,2217-2226 (1989)] of the Candida maltosa Alk1 gene can be ligatedupstream of the 5′-end and downstream of the 3′-end respectively in eachof these structural genes.

[0068] To prepare the restriction enzyme site for linking the promoterand terminator to the structural gene, a PCR technique can be utilized.The primer sequences for use in the PCR are shown in SEQ ID NO:15through NO:18. The conditions mentioned above for the case (a) can beemployed as the consitions for the PCR.

[0069] As to the promoter region, ALK1p with SalI at 5′-end and NdeI at3′-end can be prepared from SEQ ID NO:15 and NO:16 using SEQ ID NO:6 asa template. As to the terminator region, ALK1t with HindIII at 5′-endand EcoRV at 3′-end can be prepared from SEQ ID NO:17 and NO:18 usingSEQ ID NO:7 as a template. As to the vector, the vector pUTA1 (FIG. 2)prepared by modifying the marker gene from Ura3 to Ade1 using pUTU1 andCandida maltosa Ade1 gene (SEQ ID NO:21, GenBank D00855) [S. Kawai etal., Agric. Biol. Chem., Vol. 55, 59-65 (1991)]. By ligating ALK1p tothe PvuII, NdeI site of pUCNT (described in WO 94/03613) and ALK1t tothe HindIII, SspI site of the PUCNT, pUAL1 (FIG. 6) can be constructed.Then, by ligating ORF2 to the NdeI, PstI site of pUAL1, the plasmidpUAL-ORF2 (FIG. 7) can be constructed. Further, by ligating ORF3 to theNdeI, HindIII site of pUCNT-ALK1t in the course of construction of pUAL1and further ligating ALK1p, pUAL-ORF3 (FIG. 8) can be constructed.

[0070] Then, by excising ORF2, upstream promoter and downstreamterminator as a unit from the plasmid pUAL-ORF2 using EcoT22I andligating it to the PstI site of pUTA1, pUTA-ORF2 can be constructed.

[0071] In addition, by excising ORF3, upstream promoter and downstreamterminator as a unit from pUAL-ORF3 using SalI and ligating it to theSalI site of pUTA-ORF2, a plasmid pUTA-ORF23 (FIG. 9) can beconstructed.

[0072] By the above procedure, there can be constructed a geneexpression cassette for the production of a copolyester of alkanoicacids of the above-general formula (1) in the yeast Candida maltosa.

[0073] (4) Construction of a Transformant

[0074] Introduction of the polymer synthesis-associated gene expressioncassette cloning vector into a yeast can be carried out by the per seknown method. Thus, the calcium method [Lederberg. E. M. et al., J.Bacteriol, 119, 1072 (1974)] or the electroporation method [CurrentProtocols in Molecular Biology, 1, 1. 8. 4., 1994], for instance, can beemployed. A commercial transformation kit, such as Fast Track™-YeastTransformation Kit^(SM) (Geno Technology), can also be utilized.

[0075] For example, Yarrowia lipolytica CXAU1 strain [T. Iida et al.,Yeast, 14, 1387-1397 (1998)] can be used as the host. By transformingthis strain with the polymer synthesis-associated gene expressioncassette by the above transformation method, there can be constructedYarrowia lipolytica PHA1 containing pSUT-PHA1 and Yarrowia lipolyticaPHA2 containing pSUT-PHA2.

[0076] As the host, Candida maltosa CHAL [S. Kawai et al., Agric. Biol.Chem., Vol. 55, 59-65 (1991)] can also be used. By transforming thisstrain with the polymer synthesis-associated gene expression cassette bythe above transformation method, Candida maltosa CHAL containingpUTA-ORF23 can be constructed.

[0077] (5) Production of a Polyester

[0078] In the method of producing a polyester according to the presentinvention, the polyester is harvested from a culture obtained by growingthe transformant of the invention.

[0079] The production of a polyester by cultivation of a transformant ofthe invention can be carried out in the following manner. The carbonsource for use in this cultivation may be any substance that the yeastis able to assimilate. Furthermore, when the expression of the promoteris inductive, an inducer can be added as needed. There are cases inwhich the inducer serves as a principal source of carbon. Referring tonutrients other than the carbon source or sources, a medium containingsources of nitrogen, inorganic salts and other organic nutrient sourcescan be utilized. The cultivation temperature may be a temperature atwhich that strain can grow but is preferably 20° C. to 40° C. Thecultivation time is not particularly restricted but may range from about1 day to 7 days. Thereafter, the polyester can be harvested from thegrown cells or the culture broth thus obtained.

[0080] The carbon source which can be used includes carbohydrates suchas glucose, glycerol, sucrose, etc., oils and fats, fatty acids, andeven n-paraffins and the like. As said oils and fats, there can be usedrapeseed oil, coconut oil, palm oil and palm kernel oil, among others.As said fatty acids, there can be mentioned saturated or unsaturatedfatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauricacid, oleic acid, palmitic acid, linoleic acid, linolenic acid, myristicacid, etc., and derivatives of such fatty acids, e.g. their esters andsalts. The cultivation of Candida maltosa or Yarrowia lipolytica, forinstance, can be carried out using an oil or fat as the carbon source.In the case of a yeast which cannot assimilate oils and fats at all orcannot assimilate them with good efficiency, the efficiency can beimproved by adding lipase to the medium. Moreover, by transforming thelipase gene, an oil-assimilating function can be imparted.

[0081] Moreover, by using a fatty acid or n-paraffin having an oddnumber of carbon atoms as the carbon source, the proportion of theodd-number component of the carbon chain can be increased in thecopolyester of 3-hydroxyalkanoic acids of the above general formula (1).

[0082] The nitrogen source includes ammonia, ammonium salts such asammonium chloride, ammonium sulfate, ammonium phosphate, etc., peptone,meat extract, yeast extract and so on. As to said inorganic salts, therecan be mentioned, for example, potassium phosphate, potassiumdiphosphate, magnesium phosphate, magnesium sulfate and sodium chloride,etc.

[0083] As other nutrients, there can be mentioned, for example, aminoacids, such as glycine, alanine, serine, threonine, proline, etc., andvitamins such as vitamin B1, vitamin B12, biotin, nicotinamide,pantothenic acid and vitamin C, etc.

[0084] In the practice of the invention, the following techniques can beused for harvesting the polyester from the grown cells. Thus, aftercompletion of cultivation, the culture broth is centrifuged withcentrifuge to separate the cells and those cells are washed withdistilled water, methanol or the like and dried. From the dry cells, thepolyester is extracted into an organic solvent such as chloroform. Fromthis organic solvent solution containing the polyester, the cellularcomponent is removed by filtration or the like and the polyester isprecipitated by adding a poor solvent, such as methanol, hexane or thelike, to the filtrate. After removal of the supernatant by filtration orcentrifugation, the precipitated polyester is dried and recovered. Thethus-obtained polyester is analyzed by gas chromatography or nuclearmagnetic resonance spectrometry, for instance.

[0085] The method of producing a polyester according to the presentinvention, constituted as above, is capable of producing a copolyesterof 3-hydroxyalkanoic acids of the above general formula (1) with goodproductivity.

[0086] Furthermore, by the above-mentioned method which comprisesconstructing a recombinant Yarrowia lipolytica strain containing theplasmid pSUT-PHA1 or pSUT-PHA2 or a recombinant Candida maltosa straincontaining the plasmid pUTA-ORF23, for instance, and growing eitherstrain, said copolyester P(3HB-co-3HH) resulting from thecopolymerization of 3-hydroxybutyric acid of the above general formula(2) and 3-hydroxyhexanoic acid of the above general formula (3) can beproduced.

EXAMPLES

[0087] The following examples illustrate the present invention infurther detail. It should, however, be understood that the technicalscope of the invention is by no means defined by those examples.

Example 1 Polyester Synthesis-Associated Genes

[0088] (a) When Yarrowia lipolytica was Used as the Host

[0089] As polyester synthesis-associated enzyme gene, the PHA synthasegene (phac; SEQ ID NO:1) derived from Aeromonas caviae and (R)-specificenoyl-CoA hydratase gene (phaJ; SEQ ID NO:2) which converts enoyl-CoA,an intermediate in the β-oxidation pathway, to the monomeric(R)-3-hydroxyacyl-CoA [T. Fukui et al., FEMS Microbiology Letters, Vol.170, 69-75 (1999)] were used.

[0090] (b) When Candida maltosa was Used as the Host

[0091] As polyester synthesis-associated enzyme gene, this gene wasconstructed with reference to the amino acid sequences of Aeromonascaviae-derived PHA synthase and (R)-specific enoyl-CoA hydratase whichconverts enoyl-CoA, an intermediate in the β-oxidation pathway, to themonomeric (R)-3-hydroxyacyl-CoA [T. Fukui et al., FEMS MicrobiologyLetters, Vol. 170, 69-75 (1999)].

[0092]Candida maltosa is a yeast which translates the CTG codontoserine, not to leucine. Therefore, for use in Candida maltosa, CTG wasnot assigned to the leucine-specifying codon. As the codon correspondingto each amino acid, the codon with the high frequency of usage inCandida maltosa was preferentially selected. For information on thefrequency of usage of each codon, Klaus Wolf: Nonconventional Yeast inBiotechnology (published by Springer) was consulted. Specifically, ATGand TGG were assigned to the methionine-specifying codons and thetryphtophan-specifying codons, respectively. TTT or TTC was assignedalternately to the phenylalanine-specifying codons. As theleucine-specifying codons, CTC and CTG, both of which are used in DNAsequences of Aeromonas caviae, were modified to TTA and TTGrespectively, while TTA and TTG were used as such. As theisoleucine-specifying codons, ATC and ATA, both of which are used in DNAsequences of Aeromonas caviae, were modified to ATT and ATCrespectively, while ATT was used as such. As the valine-specifyingcodons, GTG and GTA, both of which are used in DNA sequences ofAeromonas caviae, were modified to GTT, while GTC and GTT were used assuch. As the serine-specifying codons, AGC, TCA and TCG, all of whichare used in DNA sequences of Aeromonas caviae, were modified to TCT,while TCC and TCT were used as such. As the proline-specifying codons,all of the corresponding codons were modified to CCA. As thethreonine-specifying codons, ACC, ACG and ACA, all of which are used inDNA sequences of Aeromonas caviae, were modified to ACT, ACC and ACCrespectively, while ATC was used as such. As the alanine-specifyingcodons, GCC, GCG and GCA, all of which are used in DNA sequences ofAeromonas caviae, were modified to GCT, GCC and GCT respectively, whileGCT was used as such. As the tyrosine-specifying codons, TAT and TACwere assigned alternatively as the tyrosine-specifying codons as used inDNA sequences of Aeromonas caviae. As stop codons, TAA was used. As thehistidine-specifying codons, CAT and CAC were assigned alternatively asthe histidine-specifying codons as used in DNA sequences of Aeromonascaviae. As the glutamine-specifying codons, all of the correspondingcodons were modified to CAA. AAT and AAC were assigned alternatively tothe corresponding asparagine-specifying codons. As the lysine-specifyingcodons, all of the corresponding codons were modified to AAA. As theasparic acid-specifying codons, all of the corresponding codons weremodified to GAT. As the glutamic acid-specifying codons, all of thecorresponding codons were modified to GAA. As the cysteine-specifyingcodons, all of the corresponding codons were modified to TGT. As thearginine-specifying codons, all of the corresponding codons weremodified to AGA. As the glycine-specifying codons, all of thecorresponding codons were modified to GGT. In Aeromonas caviae-derivedPHA synthase DNA sequence, two T nucleotides (at No. 969 and at No.1449) were modified to C so as to construct two KpnI sites. Amino acidsequence structure was not changed by these substitutions.

[0093] Thus, PHA synthase gene (ORF2; SEQ ID NO:3) and (R)-specificenoyl-CoA hydratase gene (ORF3; SEQ ID NO:4) were accordingly designed,and based on these sequences, ORF2 and ORF3 were constructed by totalsynthesis.

Example 2 Construction of a Recombinant Plasmid and a Recombinant Strain

[0094] (a) When Yarrowia lipolytica was Used as the Host

[0095] In order that the above genes may be expressed in Yarrowialipolytica, the Yarrowia lipolytica AlK3 gene promoter ALK3p (SEQ IDNO:5) (GenBank AB010390) was ligated upstream of the 5′ end of eachgene. The restriction enzyme sites necessary for linking the promoter tothe structural gene were prepared by PCR. The primer sequences used inPCR are shown in SEQ ID NO:8 through NO:14. PCR was carried out in 25cycles of 94° C.×1 min., 55° C.×2 min., 72° C.×3 min. for amplificationof the particular gene fragment. The polymerase used was Takara Shuzo'sExTaq. Regarding the promoter region, ALK3X with XbaI at 5′-end and NdeIat 3′-end and ALK3S with SacII at 5′-end and NdeI at 3′-end wereprepared from SEQ ID NO:8 and SEQ ID NO:9 or SEQ ID NO:9 and SEQ IDNO:10 respectively, using SEQ ID NO:5 as a template.

[0096] As regards phac, an about 100 bp-fragment with NdeI at 5′-end andPstI at 3′-end was prepared from SEQ ID NO:11 and SEQ ID NO:12 using SEQID NO:1 as a template. To this fragment was ligated the remainingPstI-BamHI fragment sized about 1700 bp to construct the full-lengthphac with NdeI at 5′-end and BamHI at 3′-end. As to phaJ, phaJ fragmentwith NdeI at 5′-end and KpnI at 3′-end was prepared from SEQ ID NO: 13and NO: 14 using SEQ ID NO:2 as a template.

[0097] As to the vector, the plasmid pSUTS (FIG. 1, SEQ ID NO:19) andthe plasmid pSUT6 prepared by changing the NdeI site of pSUT5 to an XbaIsite by using a linker DNA shown in SEQ ID NO:20 were used. To themulticloning site SacII, KpnI of the above pSUT6, ALK3S and phaJ wereligated to construct the plasmid pSUT-phaJ (FIG. 3). Further, ALK3X andphaC were ligated to the multicloning site XbaI, BamHT of pSUTS toconstruct a plasmid pSUT-PHA1 (FIG. 4).

[0098] Then, from the plasmid pSUT-phaJ, ALK3S, phaJ and downstreamterminator were excised as a unit using SacII and XbaI and ligated tothe SacII, XbaI site of plasmid pSUT-PHA1 to construct a plasmidpSUT-PHA2 (FIG. 5). In this manner, two kinds of plasmids forrecombination, pSUT-PHA1 and pSUT-PHA2, were constructed. By the aboveprocedure, a gene expression cassette for producing a copolyester of3-hydroxyalkanoic acids of the above general formula (1) in the yeastYarrowia lipolytica was constructed. The overall construction diagram isshown in FIG. 10.

[0099] As the host, Yarrowia lipolytica CXAU1 strain (T. Iida et al.,Yeast, 14, 1387-1397 (1998)) was used. For the introduction of theconstructed plasmid into the host, Fast Track™-Yeast-TransformationKit^(SM) (Geno Technology) was used. Transformation was carried out inaccordance with the protocol and using a selection plate (0.67 w/v %yeast nitrogen base without amino acid, 2 w/v % glucose, 24 mg/L adeninehydrochloride, 2 w/v % agar), a recombinant strain was obtained.

[0100] (b) When Candida maltosa was Used as the Host

[0101] In order that said ORF2 and ORF3 could be expressed in Candidamaltosa, the Candida maltosa AlK1 gene promoter ALK1p (SEQ ID NO:6,GenBank D00481) was ligated upstream of the 5′-end of each gene and theCandida maltosa AlK1 gene terminator ALK1t (SEQ ID NO:7) was ligateddownstream of the 3′-end. PCR was used for the preparation of therestriction enzyme sites necessary for ligating the promoter andterminator to the structural gene. The primer sequences used for PCR areshown in SEQ ID NO:15 through NO:18. The PCR was carried out in 25cycles of 94° C.×1 min., 55° C.×2 min., 72° C.×3 min. for amplificationof the particular gene fragment. The polymerase used was Takara Shuzo'sExTaq. As to the promoter region, ALK1p with SalI at 5′-end and NdeI at3′-end was prepared from SEQ ID NO:15 and NO:16 using SEQ ID NO:6 as atemplate. As to the terminator region, ALK1t with Hind III at 5′-end andEcoRV at 3′-end was prepared from SEQ ID NO:17 and NO:18 using SEQ IDNO:7 as a template. Finally, as the vectors to which ORF2 and ORF3 wereto be ligated, there were used pUTU [M. Ohkuma et al., J. Biol. Chem.,Vol. 273, 3948-3953 (1998)] which is obtained by ligating theautonomously replicating sequence (ARS) of Candida maltosa (GenBankD29758) and URA3 gene (GenBank D12720) to pUC19 and pUTA1 (FIG. 2) whichis obtained by modifying the marker gene from Ura3 to Ade1 using Candidamaltosa ADE1 gene (SEQ ID NO:21; GenBank D00855). pUTA1 was constructedby removing URA3 gene from pUTU1 with XhoI and ligating the ADE1 genefragment as excised with SalI.

[0102] ALK1p was ligated to the PvuII, NdeI site of pUCNT (described inWO94/03613) and ALK1t was ligated to the HindIII, SspI site of the pUCNTto construct pUAL1 (FIG. 6). Then, ORF2 was ligated to the NdeI, PstIsite of pUAL1 to construct a plasmid pUAL-ORF2 (FIG. 7). Moreover, inthe course of construction of pUAL1, ORF3 was ligated to the NdeI,HindIII site of pUCNT-ALK1 and, further, ALK1p was ligated to constructpUAL-ORF3 (FIG. 8).

[0103] Then, the ORF2, upstream promoter and downstream terminator wereexcised as a unit from the plasmid pUAL-ORF2 using EcoT22I and ligatedto the PstI site of pUTA1 to construct pUTA-ORF2. Furthermore, usingSalI, ORF3, the upstream promoter and downstream terminator were excisedas a unit from pUAL-ORF3 and ligated to the SalI site of pUTA-ORF2 toconstruct a plasmid pUTA-ORF23 (FIG. 9). By the above procedure, a geneexpression cassette for producing a copolyester of 3-hydroxyalkanoicacids of the above general formula (1) was constructed in the yeastCandida maltosa. The overall construction diagram is shown in FIG. 11.

[0104] As the host, Candida maltosa CHA1 (S. Kawai et al., Agric. Biol.Chem., Vol. 55, 59-65 (1991)) was used. For introduction of the plasmidconstructed into the host, Fast Track™-Yeast Transformation Kit^(SM)(Geno Technology) was used. Transformation was carried out in accordancewith the protocol and using a selection plate (0.67 w/v % yeast nitrogenbase without amino acid, 2 w/v % glucose, 24 mg/L of histidine, 2 w/v %agar), a recombinant strain was obtained.

Example 3 Productioon of P(3HB-co-3HH) Using a Recombinant Strain ofYarrowia lipolytica

[0105] The recombinant strains of Yarrowia lipolytica containing theplasmids pSUT5, pSUT-PHA1 and pSUT-PHA2 were cultured in the followingmanner. As the preculture medium, YPD medium (1 w/v % yeast-extract, 2w/v % Bacto-Pepton, 2 w/v % glucose) was used. As the polyesterproduction medium, 1/4 YP medium (0.25 w/v % yeast extract, 0.5 w/v %Bacto-Pepton) and a mineral medium (0.7 w/v % KH₂PO₄, 1.3 w/v %(NH₄)₂HPO₄, 0.5 w/v % Pro-Ex AP-12 (Banshu Condiment), 0.04 w/v %adenine, 1 ppm thiamine hydrochloride, 1 v/v % trace metal salt solution(as dissolved in 0.1 N—HCl, 8 w/v % MgSO₄.7H₂O, 0.6 w/v % ZnSO₄.7H₂O,0.9 w/v % FeSO₄.7H₂O, 0.05 w/v % CuSO₄.5H₂O, 0.1 w/v % MnSO₄.6-7H₂O, 1w/v % NaCl)) supplemented with 2 w/v % palm oil was used.

[0106] A 500-ml Sakaguchi flask containing 100 ml of the preculturemedium was inoculated with 100 μl of a glycerol stock of eachrecombinant strain and after 20 hours of culture, a 2-L Sakaguchi flaskcontaining 500 mL of the production medium was inoculated with thepreculture at the 1 v/v % level. The inoculated flask was incubated atthe cultivation temperature of 30° C. and the shaking speed of 120 rpm.The cultivation time was 24 hours for the YPD medium and 72 hours forthe mineral medium. After autoclaving, the culture broth was centrifugedto harvest cells, which were then washed with methanol and lyophilized.The dry cells were weighed.

[0107] The dry cells thus obtained were crushed and extracted with 100ml of chloroform added under stirring overnight. The extract wasfiltered to remove cells and the filtrate was concentrated to 1 to 2 mlon an evaporator. To the concentrate was added 10 ml of hexane toprecipitate the hexane-insoluble fraction.

[0108] To about 2 mg of the hexane-insoluble fraction thus obtained wereadded 500 μl of sulfuric acid-methanol mixture (15:85) and 500 μl ofchloroform and, after sealing, heated at 100° C. for 140 minutes toobtain the polyester decomposition product methyl ester. After cooling,0.3 g of sodium hydrogen carbonate was added for neutralization. To thiswas added 1 ml of diisopropyl ether, and the mixture was stirred with astirrer. This was followed by centrifuging and the organic solvent layerwas separated and analyzed for composition by capillary gaschromatography. The gas chromatograph used was Shimadzu GC-17A and thecapillary column used was GL Science's NEUTRA BOND-1 (column length 25m, column in. dia. 0.25 mm, liquid film thickness 0.4 μm). As totemperature conditions, the temperature was increased from the initiallevel of 100° C. at the rate of 8° C./min. The obtained results ofanalysis are shown in Table 1 and the chart of a sample (3) is given inFIG. 12. The NMR analysis (JEOL, JNM-EX400) and IR analysis (ShimadzuCorporation, DR-800) of the hexane-insoluble fraction obtained were alsocarried out. As an example, the result on a sample (6) are shown inFIGS. 13 and 14, respectively. TABLE 1 Culture and analysis resultAmount of Weight polymer of cells accumulated 3HH Composition SampleMedium Strain (g/L) (wt %) (mol %) (1) 1/4 YP Control 3.56 8.9 × 10⁻²(2) PHA1 3.65 1.9 × 10⁻¹ (3) PHA2 3.43 2.6 × 10⁻¹ 15 (GC) (4) MineralControl 0.15 6.7 × 10⁻² (5) PHA1 0.19 1.4 × 10⁻¹ (6) PHA2 0.17 1.8 27(NMR)

[0109] It is apparent from the above results that the copolyester P(3HB-co-3HH) can be produced by means of the yeast Yarrowia lipolytica.

[0110] It was also found that the polymer occurred, though in a smallamount, in the yeast, too.

Example 4 Production of P(3HB-co-3HH) with a Recombinant Strain ofCandida maltosa

[0111] The recombinant strain of Candida maltosa containing the plasmidpUTA1 or pUTA-ORF23 was cultured in the following manner. As thepreculture medium, YNB medium (0.67 w/v % yeast nitrogen base withoutamino acid) supplemented with 1 w/v % casamino acids and 2 w/v % palmoil was used. As the polyester production medium, YNB mediumsupplemented with 1 w/v % casamino acids and containing, as a source ofcarbon, {circumflex over (1)} 2 w/v % palm oil, {circumflex over (2)}w/v % coconut oil, {circumflex over (3)} 2 w/v % tetradecane or{circumflex over (4)} 2 w/v % hexadecane.

[0112] A 500-ml Sakaguchi flask containing 50 ml of the preculturemedium was inoculated with 100 μl of a glycerol stock of each strain andincubated for 20 hours. Then, a 2-L Sakaguchi flask containing 500 mL ofthe production medium was inoculated with the above preculture at the 10v/v % level. Culture was carried out at the cultivation temperature of30° C. and the shaking speed of 120 rpm for the cultivation time of 72hours. The resulting culture broth was autoclaved and centrifuged toharvest cells and the cells thus obtained were washed with methanol andlyophilized. The dry cells were weighed.

[0113] The dry cells thus obtained were crushed and extracted with 100ml of chloroform added under stirring overnight. The extract wasfiltered to remove cells and the filtrate was concentrated to 1 to 2 mlon an evaporator. To the concentrate was added 10 ml of hexane toprecipitate the hexane-insoluble fraction.

[0114] As a result, white precipitates were found in cultures of theplasmid pUTA-ORF23-containing recombinant strain as cultured usingcoconut oil, tetradecane and hexadecane (Table 2). The results of NMRanalysis (JEOL, JNM-EX400) of the hexane-insoluble fraction obtained inthe culture using coconut oil are shown in Table 2 and FIG. 15. TABLE 2Culture of a recombinant strain Weight of Polymer Carbon source cells(g/L) accumulated Palm oil 12.5 − Coconut oil 10.3 ++ Tetradecane 4.4 +Hexadecane 3.6 +

[0115] It is apparent from the above results that the copolyesterP(3HB-co-3HH) can be produced by means of the yeast Candida maltosa.

INDUSTRIAL APPLICABILITY

[0116] In accordance with the present invention, a copolyester of3-hydroxyalkanoic acids of the above general formula (1), which isbiodegradable and has good physical properties, can be produced by usinga yeast.

1 21 1 1785 DNA Aeromonas caviae 1 atgagccaac catcttatgg cccgctgttcgaggccctgg cccactacaa tgacaagctg 60 ctggccatgg ccaaggccca gacagagcgcaccgcccagg cgctgctgca gaccaatctg 120 gacgatctgg gccaggtgct ggagcagggcagccagcaac cctggcagct gatccaggcc 180 cagatgaact ggtggcagga tcagctcaagctgatgcagc acaccctgct caaaagcgca 240 ggccagccga gcgagccggt gatcaccccggagcgcagcg atcgccgctt caaggccgag 300 gcctggagcg aacaacccat ctatgactacctcaagcagt cctacctgct caccgccagg 360 cacctgctgg cctcggtgga tgccctggagggcgtccccc agaagagccg ggagcggctg 420 cgtttcttca cccgccagta cgtcaacgccatggccccca gcaacttcct ggccaccaac 480 cccgagctgc tcaagctgac cctggagtccgacggccaga acctggtgcg cggactggcc 540 ctcttggccg aggatctgga gcgcagcgccgatcagctca acatccgcct gaccgacgaa 600 tccgccttcg agctcgggcg ggatctggccctgaccccgg gccgggtggt gcagcgcacc 660 gagctctatg agctcattca gtacagcccgactaccgaga cggtgggcaa gacacctgtg 720 ctgatagtgc cgcccttcat caacaagtactacatcatgg acatgcggcc ccagaactcc 780 ctggtcgcct ggctggtcgc ccagggccagacggtattca tgatctcctg gcgcaacccg 840 ggcgtggccc aggcccaaat cgatctcgacgactacgtgg tggatggcgt catcgccgcc 900 ctggacggcg tggaggcggc caccggcgagcgggaggtgc acggcatcgg ctactgcatc 960 ggcggcaccg ccctgtcgct cgccatgggctggctggcgg cgcggcgcca gaagcagcgg 1020 gtgcgcaccg ccaccctgtt cactaccctgctggacttct cccagcccgg ggagcttggc 1080 atcttcatcc acgagcccat catagcggcgctcgaggcgc aaaatgaggc caagggcatc 1140 atggacgggc gccagctggc ggtctccttcagcctgctgc gggagaacag cctctactgg 1200 aactactaca tcgacagcta cctcaagggtcagagcccgg tggccttcga tctgctgcac 1260 tggaacagcg acagcaccaa tgtggcgggcaagacccaca acagcctgct gcgccgtctc 1320 tacctggaga accagctggt gaagggggagctcaagatcc gcaacacccg catcgatctc 1380 ggcaaggtga agacccctgt gctgctggtgtcggcggtgg acgatcacat cgccctctgg 1440 cagggcacct ggcagggcat gaagctgtttggcggggagc agcgcttcct cctggcggag 1500 tccggccaca tcgccggcat catcaacccgccggccgcca acaagtacgg cttctggcac 1560 aacggggccg aggccgagag cccggagagctggctggcag gggcgacgca ccagggcggc 1620 tcctggtggc ccgagatgat gggctttatccagaaccgtg acgaagggtc agagcccgtc 1680 cccgcgcggg tcccggagga agggctggcccccgcccccg gccactatgt caaggtgcgg 1740 ctcaaccccg tgtttgcctg cccaacagaggaggacgccg catga 1785 2 405 DNA Aeromonas caviae 2 atgagcgcac aatccctggaagtaggccag aaggcccgtc tcagcaagcg gttcggggcg 60 gcggaggtag ccgccttcgccgcgctctcg gaggacttca accccctgca cctggacccg 120 gccttcgccg ccaccacggcgttcgagcgg cccatagtcc acggcatgct gctcgccagc 180 ctcttctccg ggctgctgggccagcagttg ccgggcaagg ggagcatcta tctgggtcaa 240 agcctcagct tcaagctgccggtctttgtc ggggacgagg tgacggccga ggtggaggtg 300 accgcccttc gcgaggacaagcccatcgcc accctgacca cccgcatctt cacccaaggc 360 ggcgccctcg ccgtgacgggggaagccgtg gtcaagctgc cttaa 405 3 1785 DNA Artificial SequenceDescription of Artificial Sequence PHA synthase gene 3 atgtctcaaccatcttatgg tccattgttc gaagctttgg ctcattacaa tgataaattg 60 ttggctatggctaaagctca aaccgaaaga actgctcaag ccttgttgca aactaacttg 120 gatgatttgggtcaagtttt ggaacaaggt tctcaacaac catggcaatt gattcaagct 180 caaatgaattggtggcaaga tcaattaaaa ttgatgcaac acactttgtt aaaatctgct 240 ggtcaaccatctgaaccagt tattactcca gaaagatctg atagaagatt taaagctgaa 300 gcttggtctgaacaaccaat ttatgattac ttaaaacaat cctatttgtt aactgctaga 360 catttgttggcttctgttga tgctttggaa ggtgtcccac aaaaatctag agaaagattg 420 agattctttactagacaata cgtcaacgct atggctccat ctaatttctt ggctactaac 480 ccagaattgttaaaattgac tttggaatcc gatggtcaaa atttggttag aggtttggct 540 ttattggctgaagatttgga aagatctgct gatcaattaa acattagatt gactgatgaa 600 tccgcttttgaattaggtag agatttggct ttgactccag gtagagttgt tcaaagaact 660 gaattatatgaattaattca atactctcca actactgaaa ccgttggtaa aaccccagtt 720 ttgatcgttccaccattcat taataaatat tacattatgg atatgagacc acaaaactcc 780 ttggtcgcttggttggtcgc tcaaggtcaa accgttttca tgatttcctg gagaaaccca 840 ggtgttgctcaagctcaaat tgatttagat gattatgttg ttgatggtgt cattgctgct 900 ttggatggtgttgaagccgc tactggtgaa agagaagttc acggtattgg ttactgtatt 960 ggtggtaccgctttgtcttt agctatgggt tggttggccg ccagaagaca aaaacaaaga 1020 gttagaactgctactttgtt tactactttg ttggatttct cccaaccagg tgaattgggt 1080 atttttattcatgaaccaat tatcgccgcc ttagaagccc aaaatgaagc taaaggtatt 1140 atggatggtagacaattggc cgtctccttc tctttgttga gagaaaactc tttatattgg 1200 aattactatattgattctta cttaaaaggt caatctccag ttgcttttga tttgttgcac 1260 tggaactctgattctactaa tgttgccggt aaaactcata actctttgtt gagaagatta 1320 tatttggaaaatcaattggt taaaggtgaa ttaaaaatta gaaacactag aattgattta 1380 ggtaaagttaaaactccagt tttgttggtt tctgccgttg atgatcacat tgctttatgg 1440 caaggtacctggcaaggtat gaaattgttc ggtggtgaac aaagattttt attggccgaa 1500 tccggtcatattgctggtat tattaatcca ccagctgcta acaaatacgg tttctggcac 1560 aatggtgctgaagctgaatc tccagaatct tggttggctg gtgccaccca tcaaggtggt 1620 tcctggtggccagaaatgat gggttttatt caaaacagag atgaaggttc tgaaccagtc 1680 ccagccagagtcccagaaga aggtttggct ccagctccag gtcactatgt caaagttaga 1740 ttaaacccagttttcgcttg tccaaccgaa gaagatgctg cttaa 1785 4 405 DNA ArtificialSequence Description of Artificial Sequence (R)- specific enoyl-CoAhydratase gene 4 atgtctgctc aatccttgga agttggtcaa aaagctagat tatctaaaagattcggtgcc 60 gccgaagttg ctgcttttgc tgccttatct gaagatttca acccattgcacttggatcca 120 gcttttgctg ctactaccgc cttcgaaaga ccaatcgtcc atggtatgttgttagcttct 180 ttattttccg gtttgttggg tcaacaattg ccaggtaaag gttctatttatttgggtcaa 240 tctttatctt tcaaattgcc agtctttgtc ggtgatgaag ttaccgctgaagttgaagtt 300 actgctttga gagaagataa accaattgct actttgacta ctagaattttcactcaaggt 360 ggtgctttag ctgttaccgg tgaagctgtt gtcaaattgc cataa 405 51036 DNA Yarrowia lipolytica promoter ALK3p 5 ctgcagcggc gagaccggttctgggccgac tacgacgtgc ctggagggac gctccgggag 60 aatctctttg gacgggccaagatcttcccc gaccaccctg ccggacagta caagtgggaa 120 gagggggagt ttcccttgaccaagagtgac aagagtgaga acggcaatgg agtcaatgga 180 gatgagcccg ctactaagaaacaaaaaatc tgaacaagag ccggttttag tacgatacaa 240 gagccggtac gtggacatgcagctgctttt cgaacatgaa gggagcacga ccccacgtat 300 cagtattatg caagggaccagaagtggcct cggcaaaaga ttggcctcgg tcaacaaaag 360 gtcatcatat ccgtctccgcatccgtctgt acgtgaatta tgttacttgt atctttactg 420 tactggtttg gagctacgtcgccaactaat gccaaccagt cctgtggtgt gtctataggt 480 atgtaataca agtacgagtaaatgtattgt actggtgcag cacagtagat gacggagacg 540 atgaatcggt caccacccacaaacattgcc tccaaacacc gttatattgt cttactgtcg 600 tggctgagac agactcctcggggccttgta agagggggaa tgtgtgagac agatgcccac 660 aagtgaccat gcattttgtggggcaggaga aaaaccaatg tttgtgggga tagaacccat 720 caaatgaatc taaatgaactctcccaaaat gaaccactct cttcctccaa tcaaagccct 780 gcgaaatgtc ctccgtctgtttctcggacc cttagccgta cgacgccata ttacgatagc 840 ccgccacctt aatgcgtttaacttgcatgc atgcgtctgc atacagctgc atctgtcata 900 tatgcaccat ttccccacacaactgaagtt tatatatata tactgtaagg actcctgaag 960 tggcacgaac acacctgatcacagcaacat tacagtacac tactctgctc gtattttaca 1020 atactggacg aaaatg 10366 1017 DNA Candida maltosa promoter ALK1p 6 atgcatgaac aggatttaatcccaagaaaa aagtctattt tctattttca caaggaaact 60 ggaaaaacct ttttgtgttttgaagtagct ccgtaataac ctgtaaaaaa ataaattttg 120 aagatttgac ttgctgatgaaaatgctatc agtgtagctc tagacttgat actagactat 180 gatggcaaca catggtggtcaacgtgcaag acatcaccca atgagaagac tgctaaccag 240 aaaaaaaagg ggacaaaagaaaaactcgag agaaaaagtc aaattggtgt aaaattggct 300 atttttggta ctttcctaatggggaaatta attgtttaaa attccagttt ttccagagtt 360 aagatttcga ccaattatttttaatccata tgatcttcat cattatcaac ttgtgaaaaa 420 taataatcga ggtacgtttaatacgagata ttagtctacg gctatgaatg ttggatatac 480 ttcattgacg atcagaagcttgattggtta ttcaggtgca tgtgtggata taaacccaac 540 aaattatcta gcaactgtgccttccccaca ttggtcaaag aaaccctaaa gcaaattaaa 600 atctggataa ataaatcattcatttcacat tttccggtta gtataaggtt ttttaaattt 660 ttttttacag tttagccctttcaattacca aatacggtaa caatgtgctt tgtaacatgc 720 aggggatttt ctccgttgctgttttctcca catgctttta atgtgtaata aattaaaaaa 780 attacaaaga aaaaccggcatataagcatc ggagtttaca ttgttaacta actgcaaaat 840 ggcgatgttt caaatcaacaaaatttaaaa aaaccccaaa aaaaaagtat catataaatt 900 aaactcaaaa tccttttgattgcataaaat ttttaaatct cttctttttt ttctttttta 960 ctttcttatc tattctattctttttttata tatctaattc atttataaca tctggtc 1017 7 218 DNA Candida maltosaterminater ALK1t 7 atagatggat ttttcttttt tatgtgtatt tccggttaataaatgtttaa attttttttt 60 taataaaaat atttgtagtt atttatatgc aaaaaaaaaaaatattcaaa gcaatcttcc 120 tttctttctt tatctttccc ccatgctaag gtctaaaacaccacaactta aaacccaact 180 taaccgtata atactaagat caatctccaa agatgcat 2188 32 DNA Artificial Sequence Description of Artificial Sequence Primer 8gctctagact gcagcggcga gaccggttct gg 32 9 35 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 ggacacatat gcgtccagtattgtaaaata cgagc 35 10 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 10 tccccgcggc tgcagcggcg agaccggttc tgg 33 1131 DNA Artificial Sequence Description of Artificial Sequence Primer 11ggacacatat gagccaacca tcttatggcc c 31 12 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 cccagatcgt ccagattggtctgcag 26 13 31 DNA Artificial Sequence Description of ArtificialSequence Primer 13 ggacacatat gagcgcacaa tccctggaag t 31 14 29 DNAArtificial Sequence Description of Artificial Sequence Primer 14ggggtacctt aaggcagctt gaccacggc 29 15 46 DNA Artificial SequenceDescription of Artificial Sequence Primer 15 tttttcagct ggagctcgtcgacatgcatg aacaggattt aatccc 46 16 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 ccggaattcc atatgcagatgttataaatg aattagata 39 17 32 DNA Artificial Sequence Description ofArtificial Sequence Primer 17 cggaagctta tagatggatt tttctttttt at 32 1846 DNA Artificial Sequence Description of Artificial Sequence Primer 18tttttgatat cgagctcgtc gacatgcatc tttggagatt gatctt 46 19 5804 DNAArtificial Sequence Description of Artificial Sequence Plasmid pSUT5derived from E. coli/Yarrowia lipolytica 19 aggccattct cgttactgccaaaacaccac ggtaatcggc cagacaccat ggacgagtat 60 ctgtctgact cgtcattgccgcctttggag tacgactcca actatgagtg tgcttggatc 120 actttgacga tacattcttcgttggaggct gtgggtctga cagctgcgtt ttcggcgcgg 180 ttggccgaca acaatatcagctgcaacgtc attgctggct ttcatcatga tcacattttt 240 gtcggcaaag gcgacgcccagagagccatt gacgttcttt ctaatttgga ccgatagccg 300 tatagtccag tctatctataagttcaacta actcgtaact attaccataa catatacttc 360 actgccccag ataaggttccgataaaaagt tctgcagact aaatttattt cagtctcctc 420 ttcaccacca aaatgccctcctacgaagct cgagctaacg tccacaagtc cgcctttgcc 480 gctcgagtgc tcaagctcgtggcagccaag aaaaccaacc tgtgtgcttc tctggatgtt 540 accaccacca aggagctcattgagcttgcc gataaggtcg gaccttatgt gtgcatgatc 600 aagacccata tcgacatcattgacgacttc acctacgccg gcactgtgct ccccctcaag 660 gaacttgctc ttaagcacggtttcttcctg ttcgaggaca gaaagttcgc agatattggc 720 aacactgtca agcaccagtacaagaacggt gtctaccgaa tcgccgagtg gtccgatatc 780 accaacgccc acggtgtacccggaaccgga atcattgctg gcctgcgagc tggtgccgag 840 gaaactgtct ctgaacagaagaaggaggac gtctctgact acgagaactc ccagtacaag 900 gagttcctgg tcccctctcccaacgagaag ctggccagag gtctgctcat gctggccgag 960 ctgtcttgca agggctctctggccactggc gagtactcca agcagaccat tgagcttgcc 1020 cgatccgacc ccgagtttgtggttggcttc attgcccaga accgacctaa gggcgactct 1080 gaggactggc ttattctgacccccggggtg ggtcttgacg acaagggaga cgctctcgga 1140 cagcagtacc gaactgttgaggatgtcatg tctaccggaa cggatatcat aattgtcggc 1200 cgaggtctgt acggccagaaccgagatcct attgaggagg ccaagcgata ccagaaggct 1260 ggctgggagg cttaccagaagattaactgt tagaggttag actatggata tgtcatttaa 1320 ctgtgtatat agagagcgtgcaagtatgga gcgcttgttc agcttgtatg atggtcagac 1380 gacctgtctg atcgagtatgtatgatactg cacaacctgt gtatccgcat gatctgtcca 1440 atggggcatg ttgttgtgtttctcgatacg gagatgctgg gtacaagtag ctaatacgat 1500 tgaactactt atacttatatgaggcttgaa gaaagctgac ttgtgtatga cttattctca 1560 actacatccc cagtcacaataccaccactg cactaccact acaccaaaac catgatcaaa 1620 ccacccatgg acttcctggaggcagaagaa cttgttatgg aaaagctcaa gagagagaag 1680 ccaagatact atcaagacatgtgtcgcaac ttcaaggagg accaagctct gtacaccgag 1740 aaacaggcta gctcgtcgtgttcaggaact gttcgatggt tcggagagag tcgccgccca 1800 gaacatacgc gcaccgatgtcagcagacag ccttattaca agtatattca agcaagtata 1860 tccgtagggt gcgggtgatttggatctaag gttcgtactc aacactcacg agcagcttgc 1920 ctatgttaca tccttttatcagacataaca taattggagt ttacttacac acggggtgta 1980 cctgtatgag caccacctacaattgtagca ctggtacttg tacaaagaat ttattcgtac 2040 gaatcacagg gacggccgccctcaccgaac cagcgaatac ctcagcggtc ccctgcagtg 2100 actcaacaaa gcgatatgaacatcttgcga tggtatcctg ctgatagttt ttactgtaca 2160 aacacctgtg tagctccttctagcattttt aagttattca cacctcaagg ggagggataa 2220 attaaataaa ttccaaaagcgaagatcgag aaactaaatt aaaattccaa aaacgaagtt 2280 ggaacacaac cccccgaaaaaaaacaacaa acaaaaaacc caacaaaata aacaaaaaca 2340 aaataaatat ataactaccagtatctgact aaaagttcaa atactcgtac ttacaacaaa 2400 tagaaatgag ccggccaaaattctgcagaa aaaaatttca aacaagtact ggtataatta 2460 aattaaaaaa cacatcaaagtatcataacg ttagttattt tattttattt aataaaagaa 2520 aacaacaaga tgggctcaaaactttcaact tatacgatac ataccaaata acaatttagt 2580 atttatctaa gtgcttttcgtagataatgg aatacaaatg gatatccaga gtatacacat 2640 ggatagtata cactgacacgacaattctgt atctctttat gttaactact gtgaggcatt 2700 aaatagagct tgatatataaaatgttacat ttcacagtct gaacttttgc agattaccta 2760 atttggtaag atattaattatgaactgaaa gttgatggca tccctaaatt tgatgaaaga 2820 tgaaattgta aatgaggtggtaaaagagct acagtcgttt tgttttgaga taccatcatc 2880 tctaacgaaa tatctattaaaaatctcagt gtgatcatga gtcattgcca tcctggaaaa 2940 tgtcatcatg gctgatatttctaactgttt acttgagata aatatatatt tacaagaact 3000 tcccttgaaa ttaatttagatataaaatgt ttgcgggcaa gttactacga ggaataaatt 3060 atatctgttg actagaagttatgaacattc agtatatatg cacatataat aaccaacttc 3120 ggccctttcg tctcgcgcgtttcggtgatg acggtgaaaa cctctgacac atgcagctcc 3180 cggagacggt cacagcttgtctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 3240 cgtcagcggg tgttggcgggtgtcggggct ggcttaacta tgcggcatca gagcagattg 3300 tactgagagt gcaccatacgcgcgctatag ggcgaattgg agctccaccg cggtggcggc 3360 cgctctagaa ctagtggatcccccgggctg caggaattcg atatcaagct tatcgatacc 3420 gtcgacctcg agggggggcccggtacccag cttttgtccc tgcgcgctat gcggtgtgaa 3480 ataccgcaca gatgcgtaaggagaaaatac cgcatcaggc gctgcattaa tgaatcggcc 3540 aacgcgcggg gagaggcggtttgcgtattg ggcgctcttc ctaggcaatt aacagatagt 3600 ttgccggtga taattctcttaacctcccac actcctttga cataacgatt tatgtaacga 3660 aactgaaatt tgaccagatattgttgtaaa tagaaaatct ggcttgtagg tggcaaaatc 3720 ccgtctttgt tcatcaattccctctgtgac tactcgtcat ccctttatgt tcgactgtcg 3780 tatttcttat tttccatacatatgcaagtg agatgcccgt gtcctcctcg ctcactgact 3840 cgctgcgctc ggtcgttcggctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3900 ggttatccac agaatcaggggataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3960 aggccaggaa ccgtaaaaaggccgcgttgc tggcgttttt ccataggctc cgcccccctg 4020 acgagcatca caaaaatcgacgctcaagtc agaggtggcg aaacccgaca ggactataaa 4080 gataccaggc gtttccccctggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4140 ttaccggata cctgtccgcctttctccctt cgggaagcgt ggcgctttct caatgctcac 4200 gctgtaggta tctcagttcggtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4260 cccccgttca gcccgaccgctgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4320 taagacacga cttatcgccactggcagcag ccactggtaa caggattagc agagcgaggt 4380 atgtaggcgg tgctacagagttcttgaagt ggtggcctaa ctacggctac actagaagga 4440 cagtatttgg tatctgcgctctgctgaagc cagttacctt cggaaaaaga gttggtagct 4500 cttgatccgg caaacaaaccaccgctggta gcggtggttt ttttgtttgc aagcagcaga 4560 ttacgcgcag aaaaaaaggatctcaagaag atcctttgat cttttctacg gggtctgacg 4620 ctcagtggaa cgaaaactcacgttaaggga ttttggtcat gagattatca aaaaggatct 4680 tcacctagat ccttttaaattaaaaatgaa gttttaaatc aatctaaagt atatatgagt 4740 aaacttggtc tgacagttaccaatgcttaa tcagtgaggc acctatctca gcgatctgtc 4800 tatttcgttc atccatagttgcctgactcc ccgtcgtgta gataactacg atacgggagg 4860 gcttaccatc tggccccagtgctgcaatga taccgcgaga cccacgctca ccggctccag 4920 atttatcagc aataaaccagccagccggaa gggccgagcg cagaagtggt cctgcaactt 4980 tatccgcctc catccagtctattaattgtt gccgggaagc tagagtaagt agttcgccag 5040 ttaatagttt gcgcaacgttgttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 5100 ttggtatggc ttcattcagctccggttccc aacgatcaag gcgagttaca tgatccccca 5160 tgttgtgcaa aaaagcggttagctccttcg gtcctccgat cgttgtcaga agtaagttgg 5220 ccgcagtgtt atcactcatggttatggcag cactgcataa ttctcttact gtcatgccat 5280 ccgtaagatg cttttctgtgactggtgagt actcaaccaa gtcattctga gaatagtgta 5340 tgcggcgacc gagttgctcttgcccggcgt caatacggga taataccgcg ccacatagca 5400 gaactttaaa agtgctcatcattggaaaac gttcttcggg gcgaaaactc tcaaggatct 5460 taccgctgtt gagatccagttcgatgtaac ccactcgtgc acccaactga tcttcagcat 5520 cttttacttt caccagcgtttctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5580 agggaataag ggcgacacggaaatgttgaa tactcatact cttccttttt caatattatt 5640 gaagcattta tcagggttattgtctcatga gcggatacat atttgaatgt atttagaaaa 5700 ataaacaaat aggggttccgcgcacatttc cccgaaaagt gccacctgac gtctaagaaa 5760 ccattattat catgacattaacctataaaa ataggcgtat cacg 5804 20 10 DNA Artificial SequenceDescription of Artificial Sequence linker DNA 20 tactctagag 10 21 1820DNA Candida maltosa misc_feature (538)..(1413) Ade1 21 gatccccttcttcaaacctt taaatgacat tgtttcgttt ctctatgttt ggtatcggtt 60 cttcttcttcttcaaaaaaa aggggggcac tattcaaaaa aaaatattat aacagtatga 120 tttttttccctctcccgtcg attgaggttt tttttttctc tttcgtcttg gtcttttgct 180 tttcactccaaaaatggaaa cacgcgcggc tcaactcgaa atccgtgatc aaaaaaataa 240 aggctgtgagtttcgagcca ataattatga attagtggta ttttttttaa agataaataa 300 tcaagaatcgcattagggag acgaatatgc gttattcaaa taaaaagaca attcttttag 360 ggtagcatttcccttcaagt tcatcccaca tgtacattaa tgtcaatgat gtcgcagaag 420 ttaaattagcagaagaaaaa aaaaatgtga attactccga gtcaactctt ctttctcttc 480 ttctttttcttctttatcac cataatcacc accaccacca ccaccaccag ctcccagatg 540 acttcaactaacttagaagg aactttccca ttgattgcca aaggtaaagt cagagatatt 600 taccaagttgacgacaacac tcttttattc gttgctactg atagaatttc cgcatacgat 660 gtgattatgtctaatggtat cccaaataaa ggtaaaatct taaccaaatt gtctgaattc 720 tggtttgatttcttgccaat tgaaaaccat ttaatcaaag gagacatttt ccaaaaatat 780 cctcaactagaaccatatag aaaccaattg gaaggcagat ccttacttgt tagaaaattg 840 aaattgatccctcttgaagt tattgttaga ggttacatca ccggttccgg ctggaaagaa 900 taccaaaaatctaaaaccgt ccacggtatt cctattggtg atgtggttga atcacaacaa 960 atcactcctatcttcacccc atccactaaa gcagaacaag gtgaacatga tgaaaatatc 1020 accaaagaacaagctgacaa gattgttgga aaagaattat gtgatagaat tgaaaaaatt 1080 gctattgatttgtacaccaa agccagagat tacgctgcca ctaaaggaat tattatcgct 1140 gatactaaatttgaatttgg tttagatggt gacaacatcg ttcttgttga cgaagtttta 1200 actccagattcttccagatt ctggaatgct gctaaatacg aagttggtaa atctcaagac 1260 tcttacgataaacaattttt gagagattgg ttaacttcta atggtgttgc tggtaaagat 1320 ggtgttgctatgcctgaaga cattgtcact gaaaccaaga gcaaatacgt tgaagcttac 1380 gaaaatttaactggtgacaa atggcaagaa taaattaagg atatctatta ttaaagcttt 1440 ctatttatcccaaactttcg tagtattttc tgacatgttc agatgttttt actttatctt 1500 tcctgaaatttttgatttct aaccgactct tgcatgtagc tcttgataat gcaacatatg 1560 cttgaccattagcaaaactt ctacctaaat ctattttgac tctgtccaaa gtttgacctt 1620 gagctttgtggatcgacatc gcccacgaca agatcatttg gtttgttttt atggtgggtt 1680 attggcacttggtgcaactg atggtttaac tttggaagag gctaagaaat tgaagacttg 1740 gaatgaagaacgtgcatctg atttcaaatt gggtgaagaa ttgacttata cttgttataa 1800 aatgtatcatgatgttgatc 1820

1. A transformant wherein at least one kind of gene expression cassettecomprising a polyester synthesis-associated enzyme gene has beenintroduced into a yeast.
 2. The transformant according to claim 1wherein the polyester is a copolymer resulting from the copolymerizationof 3-hydroxyalkanoic acids of the following general formula (1);

in the formula, r represents an alkyl group.
 3. The transformantaccording to claim 1 or 2 wherein the polyester is copolyesterP(3HB-co-3HH) resulting from the copolymerization of 3-hydroxybutyricacid of the following formula (2) and 3-hydroxyhexanoic acid of thefollowing formula (3);


4. The transformant according to any of claims 1 to 3 wherein the yeastbelongs to any of the genera Aciculoconidium, Ambrosiozyma, Arthroascus,Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma,Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora,Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis,Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium,Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum,Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia,Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora,Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium,Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma,Nadsonia, Nakazawaea, Nematospora, Ogataea, Oosporidium, Pachysolen,Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula,Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia,Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces,Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus,Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces,Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon,Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia,Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus,Zygosaccharomyces, Zygowilliopsis and Zygozyma.
 5. The transformantaccording to any of claims 1 to 4 wherein the yeast is Yarrowialipolytica.
 6. The transformant according to any of claims 1 to 4wherein the yeast is Candida maltosa.
 7. The transformant according toany of claims 1 to 6 wherein a polyester synthesis-associated enzymegene expression cassette comprises a promoter and a terminator, saidpromoter and said terminator functioning in a yeast.
 8. The transformantaccording to claim 7 wherein the promoter and terminator are derivedfrom Yarrowia lipolytica.
 9. The transformant according to claim 7 or 8wherein the promoter is derived from Yarrowia lipolytica ALK3.
 10. Thetransformant according to claim 7 or 8 wherein the terminator is derivedfrom Yarrowia lipolytica XPR2.
 11. The transformant according to claim 7wherein the promoter and terminator are derived from Candida maltosa.12. The transformant according to claim 7 or 11 wherein the promoter isderived from Candida maltosa ALK1.
 13. The transformant according toclaim 7 or 11 wherein the terminator is derived from Candida maltosaALK1.
 14. The transformant according to any of claims 1 to 13 whereinthe polyester synthesis-associated enzyme gene is derived from Aeromonascaviae.
 15. The transformant according to any of claims 1 to 13 whereinthe polyester synthesis-associated enzyme gene is a PHA synthase genederived from Aeromonas caviae or a PHA synthase gene and (R)-specificenoyl-CoA hydratase gene.
 16. The transformant according to claim 15wherein said PHA synthase gene has the sequence represented by SEQ IDNO:3 and the (R)-specific enoyl-CoA hydratase gene has the sequencerepresented by SEQ ID NO:4.
 17. A method of producing a polyester usingthe transformant according to any of claims 1 to 16 which comprisesgrowing said transformant and harvesting a polyester from the resultingculture.
 18. A polyester synthesis-associated enzyme gene which ismodified from at least one gene code CTG to TTA, TTG, CTT, CTC or CTA.19. The polyester synthesis-associated enzyme gene according to claim 18which codes for an enzyme derived from a bacterium.
 20. The polyestersynthesis-associated enzyme gene according to claim 19 wherein saidbacterium is Aeromonas caviae.
 21. The polyester synthesis-associatedenzyme gene according to claim 20 wherein the enzyme gene derived fromAeromonas caviae is a PHA synthase gene or a (R)-specific enoyl-CoAhydratase gene.
 22. The polyester synthesis-associated enzyme geneaccording to claim 21 wherein said PHA synthase gene has the sequencerepresented by SEQ ID NO:3.
 23. The polyester synthesis-associatedenzyme gene according to claim 21 wherein said (R)-specific enoyl-CoAhydratase gene has the sequence represented by SEQ ID NO:4.